Three hamster clones (clones 1, 2 and 3) were isolated from a genomic library constructed from a homozygote rapid acetylator using a cDNA (hamAT-101) of a monomorphic acetyltransferase (AT-I) as a probe. Clone 1 (13 kbp) was found to contain a gene corresponding to AT-I. The entire coding region was located in an exon and completely identical to that of AT-I cDNA. Clones 2 and 3 (14.5 and 15 kbp) each contained identical information to the AT-I-related protein (AT-B protein). The intronless coding region shared 83.7% of sequence similarly to the AT-I cDNA, and its length was identical to that of the AT-I cDNA. Clone 2 also included a nucleotide sequence identical to the 3'-portion of the AT-I gene, which is located 5'-upstream of the AT-B gene. Restricted fragment lengths of clone 3, which encompassed the entire coding region was expressed in COS-1 cells. The expressed protein migrated at a position identical to that of AT-II purified from a hamster liver on Western blots. AT-B-expressed protein catalysed acetyl CoA-dependent N-acetylation of 2-aminofluorene and p-aminobenzoic acid, but had marginal activities for O-acetylation of 2-N-hydroxyamino-6-methyl-6-methyldipyrido[1,2-a:3',2'-d]imidazole and N-hydroxyarylacetamide-dependent N-acetylation of 4-aminoazobenzene. These results are in good agreement with the data of AT-II purified from hamster livers, indicating that the AT-B gene encodes a polymorphic acetyltransferase (AT-II) in hamsters. Although the AT-II protein was undetectable in slow acetylators, specific mRNA, hybridizing with a selective oligonucleotide probe for the AT-II gene (AT-B), was detected in livers of both homozygous acetylators. Analysis of genomic DNA of a homozygous slow phenotype hamster indicates that AT-II DNA from the slow phenotype has a point mutation which causes premature termination at the 243th (Arg to stop codon) position of the deduced amino acid sequence. PCR-RFLP analysis further confirmed that the point mutation conferred a defective AT-II protein in slow phenotype hamsters.